Recent studies suggest that IGF-IR activity and p53 function may be closely related. These studies demonstrate that activation of caspase 9 is a critical downstream effect of p53 action and that caspases 9 activation is required for p53 dependent cell death. Caspase 9 is also a substrate of Akt, a kinase activated by IGF-I. Akt phosphorylation of caspase 9 represses caspase 9 activity. Thus, IGF-I and Akt may inhibit 953 induced cell death via modulation of caspase. Our lab has key tools available to characterize the importance and activity of IGF-IR and Akt in the inhibition of p53 dependent cell death. We hypothesize that blocking IGF-I induced Akt activity will augment cell death mediated by the p53 downstream targets, caspase 9 and Apaf-1. In doing so, we expect to reconstitute sensitivity to DNA damage, a characteristic associated with p53 dependent cell death, in breast tumor cells that overexpress Mt p53 or mdm-2. We will achieve this goal in four Aims. Aim 1 will determine if p53 induced apoptosis is associated with Apaf-1/Caspase 9 /Cytochrome C complex formation in MCF-7 breast cancer cells. Aim 2 will determine if IGF-IR induced Akt phosphorylates caspase 9 and if caspse 9 phosphorylation inhibits p53- induced apoptosis. Aim 3 will determine if inducible expression if caspase 9 and Apaf-1 results in cell death after irradiation or etopside treatment of MCF-7 cells over expressing either Mt p53 or m.m.-2. Cells will also be treated with IGF-I To confirm that IGF-IR survival effects occur through inhibition of caspases 9 activity. Aim 4 3will identify agents that cause caspase 9 cleavage independent of Wt 953. Clearly, if treatments are identified which result in caspase cleavage without the requirement of p53 this would be of great clinical benefit in predicting efficacy of agents in women with breast tumors express Mt p53 or overexpress m.m.-2. If caspase 9 can function independently of p53 this would suggest that cancer cells deficient in Wt p53 may ultimately be resensitized to DNA damaging agents by targeting downstream caspase 9. The loss of p53 function or increased m.m.-2 expression is frequently observed in human breast tumors and advances in the understanding of how these aberrations affect response to treatment are likely to have broad implications to breast cancer patients. Inhibition of IGF-I Or Akt action may well augment the efficiency of these new therapeutic strategies. Enhancing responses through p53 downstream targets and inhibiting IGF-I survival properties on breast cancer cells are cornerstone to these advances.